Effect of a Synbiotic Formula for reducing C. jejuni in infected broilers. EURL - Campylobacter workshop

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1 Effect of a Synbiotic Formula for reducing C. jejuni in infected broilers EURL - Campylobacter workshop Dr. Giuliano Garofolo Istituto Zooprofilattico Sperimentale dell Abruzzo e del Molise G. Caporale Teramo, 29 September 1 October 2014 First Hotel Linné, Uppsala, Sweden

2 We are here

3 Background 30% The public health benefits of controlling Campylobacter in primary broiler production are expected to be greater than control later in the chain as bacteria may also spread from farms to humans by other pathways than broiler meat EFSA Journal 2011; 9(4):2105

4 INTERVENTION AT THE PRIMARY PRODUCTION Reduction of C. jejuni Feed additive ANTIMICROBIAL ACTIVITY vs. C. jejuni SURVIVAL TO GI CONDITIONS SURVIVAL TO INDUSTRIAL PROCESSING SAFETY ASSESSMENT Probiotic Microencapsulated B. longum subsp. longum PCB133 strain as probiotic in order to obtain an intake of 10 9 CFU of PCB133/day Prebiotic plus 0.2 % XOS prebiotic

5 Experimental set up 160 Ross broiler 1 day old chicks from the local hatchery A: Conventional feed Chicks were orally inoculated with C. jejuni M1(10 5 CFU/bird) C: Symbiotic blend from day 1 B: Symbiotic blend from day 14 D: Probiotic strain from day 14

6 Chickens growth condition Age 0 10 day day Conv. Feed Starter Fattening I Age 1 day 4 day 7 day 10 day 13 day 16 day Temp. 33 C 32 C 31 C 30 C 29 C 28 C Age I week II week - end Day light 23 h / 1h 18 h / 6h day day Fattening II Finishing 19 day 22 day 25 day 27 C 26 C 25 C 28 day 24 C 31 day 23 C 34 day 22 C day 21 C

7 Experimental set up 1 d 5d 10d 20d 30d 39d Challenge Check infection I Sampling II Sampling III Sampling IV Sampling

8 Methods 4 chickens were slaughtered at day 5 to check C.jejuni colonization 9 chickens were tested each sampling Detection and enumeration of Campylobacter spp. of Campylobacter spp. from cecum contents (ISO :2006) Real Time qpcr cecum contents 16s DNA (Bifidobacterium spp., B. longum subsp. longum, Campylobacter spp.) - hipo gene (C. jejuni)

9 Results 1. To verify in vivo activity of the B. longum subsp. longum PCB133 with Xylooligosaccharides (XOS) to reduce the cecal concentration of C. jejuni 7 C. jejuni M1mean log10 CFU g Group A Group B Group C Group D 0 Group A Group B Group C Group D Day 40 Group C had lower counts by 3 log10 CFU with the Group A (control animals) All of the treated animals had lower caecal Campylobacter jejuni

10 Group C symbiotic additive from day 1 Log10 CFU Group C day 20 day 30 day 39 day The variability between groups of Campylobacter load in caeca was calculated using the analysis of variance (ANOVA). Sampling Comparison p-value 1 A~B 0,031 1 A~C 0,020 1 A~D 0,135 2 A~B 0,156 2 A~C 0,062 2 A~D 0,266 3 A~B 0,234 3 A~C 0,065 3 A~D 0,199 4 A~B 0,055 4 A~C 0,004 4 A~D 0,447

11 Results (Group A vs Group C) Log 10 CFU Group A Log10 CFU Group C ,13 7, ,51 2,9 Group A Group B Group C Group D day 20 day 30 day 39 day

12 Bifidobacterium Real time Q PCR Bifidobacterium spp. Bifidobacterium longum log10(cfu/g) 9,0 8,0 7,0 6,0 5,0 4,0 3,0 GrpA GrpB GrpC GrpD Treatments ST1 ST2 ST3 ST4 log10(cfu/g) 8,0 7,0 6,0 5,0 4,0 3,0 2,0 1,0 0,0 GrpA GrpB GrpC GrpD Treatments ST1 ST2 ST3 ST4 Statistical significances were confirmed by singularly comparing the different treatments with the control group using Duncan

13 Campy Real time Q PCR Campylobacter spp. Campylobacter jejuni 10,0 10,0 9,0 9,0 log10(cfu/g) 8,0 7,0 6,0 5,0 ST1 ST2 ST3 ST4 log10(cfu/g) 8,0 7,0 6,0 5,0 4,0 ST1 ST2 ST3 ST4 4,0 3,0 GrpA GrpB GrpC GrpD Treatments GrpA GrpB GrpC GrpD Treatments

14 Conclusion Agreement between Microbiogical and molecular methods The results underlined the effectiveness of the synbiotic product in reducing Campylobacter jejuni in infected chickens (3 log10 of reduction). Considering the high infective dose in 1-day-old chickens the product showed the effect only with a lifelong supplementation. An interesting outcome concerned the high number of endogenous bifidobacteria in control group that are ineffective in reducing C. jejuni load in caeca compared to the probiotic B. longum subsp. longum PCB133. Denaturing Gradient Gel Electrophoresis (DGGE) to check gut microbiota are in progress

15 Conclusion Reducing the numbers of Campylobacter in the intestines at slaughter by 3 log10-units, would reduce the public health risk by at least 90%. It would be possible to have an integrate approach to achieve this result plus

16 Next Intervention measures Field experiments Will be performed in a conventional farm using the symbiotic additive from the first day of the crop cycle

17 Acknowledgements NRL Campylobacter Teramo - Italy Department of Agricultural Science (DipSA), University of Bologna

18 Thank you